This document describes an experiment to localize disease genes for inherited eye disorders through linkage analysis. It involves genotyping DNA samples from a pedigree using PCR and microsatellite markers. The results will be analyzed using linkage analysis software to calculate LOD scores and determine if marker loci are co-segregating with the disease trait, helping to map the location of the disease gene. Equipment and reagents needed for PCR, electrophoresis, and linkage analysis are listed.

1. EXPERIMENT
TRANSFORMATION OF PERIPHERAL BLOOD LYMPHOCYTES (PBLs)
BY EPSTEIN-BARR VIRUS
Introduction
Epstein-Barr virus (EBV) is a double stranded DNA virus that infects human B lymphocytes. It
enters the lymphocyte through the CD23 receptor and transforms them into continuously
growing lymphoblastoid cell lines which can provide a constant source of DNA.
Equipment and Materials
Laminar air flow hoods
Bench top centrifuges
15 and 50 ml polypropylene centrifuge tubes
Cryovials
Tissue Culture Medium: RPMI-1640
Fetal Calf Serum (FCS)
Histopaque-1077 (Sigma Chem. Co.)
Dimethyl Sulphoxide (DMSO)
ß-mercapto ethanol (BME)
Cyclosporin
L- Glutamine
Na-Pyruvate
Penicillin-G
Streptomycin
Trypan Blue
Sodium bicarbonate
Hydrochloric acid
EBV supernatant
RPMI-1640 Medium
Add 103.9 g powder medium to 8 liter deionized water, add 20 g sodium bicarbonate. Allow the
solids to dissolve completely by constant stirring. Adjust pH to 7.2 with HCl. Make up the
volume to 10 liter. Filter the medium through 0.2 µm membrane filter and dispense 450 ml into
sterile bottles (500 ml) using sterile tissue culture techniques. Store the filtered medium at 4oC.
GPPS
L-Glutamine: 2.922 g
Na-Pyruvate: 1.1004 g
Penicillin-G: 0.300842 g
Streptomycin: 0.5 g
Dissolve in deionized water and make up the volume up to 100 ml. Filter the solution through
0.2µm membrane filter and store at -20oC until needed.
1

2. Supplemented Culture Medium (10% FCS)
To the filtered RPMI-1640 medium (450 ml) add 50 ml FCS and 5 ml GPPS solution. Store at
4oC.
Wash Medium (1.0% FCS)
To the filtered RPMI-1640 medium (450 ml) add 5 ml FCS and 5 ml GPPS. Store at 4oC.
Transformation Medium
To 500 ml supplemented culture medium add 500 µl of ß-mercaptoethanol solution (0.05 M
stock solution) and 500 µl cyclosporin (200 µg/ml stock). Store at 4oC.
Freezing Mix
Take equal volumes of RPMI-1640 medium and FCS (90 ml each). Add 10% (20 ml) sterile
DMSO. Store at 4oC.
Trypan Blue Solution
Dissolve 0.016 g of trypan blue in 10 ml normal saline solution (0.9%). Filter through 0.5µm
filter paper. Store at room temperature.
Procedure
1. Preparation of EBV supernatant
Culture B95-8 marmoset cells (1-5 X 106 cells) in tissue culture medium. Grow the cells
at 37oC up to a concentration of approximately 5 X 106 cell/ml. Expand the cultures and
when cell density reaches 5 X 106-107/ml and the media changes its color (turns yellow)
then place the flask at 34oC for a week. Centrifuge the contents of the flask at 300 g for
10 min. Filter the supernatant through a 0.45 µm membrane filter under sterile
conditions using a membrane filtration assembly. Aliquot into cryovials and store at
-70oC.
2. Sample collection
Collect blood samples (3-5 ml) in vacutainers containing acid citrate dextrose (ACD)
under sterile conditions (i.e by puncturing the vacutainer rubber top with syringe needle
and not by opening the tube outside the laminar air flow hood).
3. Isolation of peripheral blood lymphocytes
Take 3 ml Histopaque in a 15 ml falcon tube. Gently layer 5 ml blood on Histopaque
and centrifuge at 400g for 20 minutes. Take the buffy layer at the interphase of the
plasma and Histopaque into a 15 ml Falcon tube containing 10 ml wash medium. Mix
and pellet cells by centrifugation at 300 g for 10 minutes. Discard the supernatant and
wash the cell pellet two times with the 5 ml wash medium.
2

3. 4. Cell counting and trypan blue exclusion test
Resuspend pellet in 1 ml wash medium. Take 10 µl of the cell suspension and mix it with
(equal volume) 10 µl of trypan blue solution. Count the cells using a haemacytometer.
Count the number of live and dead cells (which are stained blue) in 25 squares in the
center of the counting chamber. Calculate the percentage viability by using the following
formula:
number of live cells/total number of cells X 100.
Calculate the number of live cells per ml by using the following formula:
live cell count X dilution factor (2.0) X 104.
5. Transformation with EBV
Transfer 2-3 X 106 live cells to a 25 cm2 culture flask containing 3 ml transformation
medium and 1 ml EBV supernatant. Incubate the flasks at 37oC in a humidified
atmosphere of 95% air and 5% CO2. Tighten the flask caps after 24 hours and leave them
until visible colonies are formed. Subsequently loosen the culture flask cap by half a turn
and observe the cultures periodically. Add supplemented culture medium and subculture
into culture flasks when required (medium turns yellow and the cell density increases).
6. Cryopreservation of cultures
Check the viability of the cell culture by trypan blue exclusion test. Take an appropriate
volume of cell suspension for approximately 5 X 106 cells with a viability of 90% or
above. Pellet the cells by centrifugation at 300 g for 10 minute. Mix the pellet with 1 ml
freezing mix and transfer into 1 ml cryovials. Place the vials in a polystyrene box and
keep at -70oC overnight so that the temperature drop is gradual (1oC per minute.). For
long term storage transfer the vials to liquid nitrogen cryostorage system.
7. Revival of frozen cultures
Whenever required, revive the frozen cells by transferring the contents of the frozen vial
into a 15 ml Falcon tube containing 10 ml wash medium. Mix the suspension and pellet
by centrifugation at 300 g for 10 minutes. Wash the pellet twice with 5 ml wash medium.
Re-suspend the washed pellet in 5 ml cell culture medium, transfer it into 25 cm2 tissue
culture flask and grow the culture at 37oC in a humidified incubator with 5 % CO2 in air
mixture.
3

4. EXPERIMENT
PREPARATION OF GENOMIC DNA FROM EBV TRANSFORMED
LYMPHOBLASTOID CELL LINES
Introduction
Mammalian DNA is extracted by organic methods that involve digestion of cells with proteinase
K in the presence of EDTA and a detergent such as SDS, followed by extraction with phenol.
Equipment and Materials
Refrigerated centrifuges
15 and 50 ml Centrifuge tubes
Waterbaths
1M Tris, pH 8.0 1 liter
• Trizma Base 121.1 g
Dissolve Tris in~800ml dH2O ,adjust pH to 8.0 with concentrated HCl; q.s to 1L dH2O and filter
through 0.4µm filter paper .Store at room temperature (0.1M & 10mM Tris are also prepared
from this stock).
0.5M EDTA, pH 8.0 1 liter
• EDTA 186.15 g
Dissolve 186.15g of EDTA in700ml dH2o and adjust pH 8.0 with 4N NaOH. q.s to 1L with
dH2O
STE (Saline Tris EDTA) pH 8.0 1 liter
• 3M NaCl 33.3 ml 100 mM
• 1M Tris, pH8.0 50.0 ml 50 mM
• 0.5M EDTA pH 8.0 2.0 ml 1 mM
Measure reagents mix and q.s to 1L with dH2o place at room temperature.
10% SDS (Laryl Sulfate) 100 ml
• SDS 10 g
Dissolve in dH2O, filter and store at room temperature.
Proteinase K (20 mgml) 1ml
• Proteinase K 20 mg
Dissolve in 1ml of dH2O & store at -20°C
4

5. Phenol Equilibration
• Phenol 1 kg
• 1.0M Tris, pH 8 2 liter
• 0.1M Tris, pH 8 2 liter
• 8-hydroxy quinoline 1g
• β-mercaptoethanol 2 ml
Add 8-hydroxy quinoline to melted distilled phenol. Extract with equal volume of 1M tris, then
extract with an equal volume of 0.1M tris, until pH of aqueous layer is 8.0, finally add 100 ml
0.1M Tris containing 0.2% β-mercaptoethanol.
RNAse (10mgml) 1ml
• RNAse A 10 mg
• 10mMTris, pH 7.5 1 ml
Dissolve in 1ml of (10mM) Tris, pH 7.5 and store at -20°C
Chloroform:isoamy alcohol (24:1) 500 ml
• Chloroform 480 ml
• Isoamyl alcohol 20 ml
Mix the two solution & store at 4°C
Isopropanol (extra pure)
Store at 4°C
70% ethanol (freshly distilled)
Store at 4°C
TE (Tris EDTA) 500ml Final conc
• 1MTris 5 ml 10 mM
• 0.5M EDTA 1 ml 1 mM
Mix and raise the volume to 500ml with dH2O, store at room temperature.
5

6. Procedure
1. Gently vortex PBMCs obtained from 20 ml blood (buffy coat) or EBV transformed
cells (approximately 5x107 in a sterile 50 ml Falcon tube).
2. Add 19 ml 0f STE buffer followed by 1 ml of 10%SDS (drop wise while gently
vortexing), add 20 µl of 20 mg/ml Proteinase-K (Sigma), Incubate samples over-night
in a shaking water bath at 55°C.
3. On the following day extract with an equal (20 ml) volume of equilibrated phenol, pH
8, mix gently on ice for 10 minutes and place on ice for 10 minutes, centrifuge at
3200 rpm for 40 minutes at 4°C, remove the aqueous layer carefully with cut tip.
4. Extract with an equal volume of ChloroformIsoamylalcohol (24:1), mix gently on ice
for 10 minutes and place on ice for 10 minutes and centrifuge again at 3200 rpm for
40 minutes at 4°C, remove aqueous layer in separate tubes.
5. Precipitate nucleic acids by adding one tenth volume of 10 M ammonium acetate
followed by 2 volumes of absolute ethanol (or an equal volume of Isopropanol) and
store after mixing at -20°C overnight or at -70°C for 15 minutes. Centrifuge at 3200
rpm for 90 minutes at 4°C, decant supernatant.
6. Add 5 ml of 70% ethanol (tap pellet so it floats) and again centrifuge at 3200 rpm for
40 minutes, decant supernatant and vacuum dry for 10 minutes.
7. Add 1 ml of TE and incubate at 37°C in a shaking water bath, for one hour to
resuspend the pellet.
8. After one hour add 10 µl of RNaseA (10 mg/ml, Sigma) to digest RNA and incubate
at 37°C for 2 hours in a shaking water bath.
9. Add 50 µl of 10% SDS and 5 µl Proteinase-K (Sigma) and incubate at 55°C in a
shaking water bath for 1 hour (after incubation sample may be stored overnight at
4°C).
10. Add 4 ml TE and extract with 5 ml of equilibrated phenol, pH 8 and mix gently on ice
for 10 minutes and place on ice for 10 minutes. Centrifuge at 3200 rpm for 40
minutes, remove the aqueous layer carefully with cut tip and add 2 ml of TE to
remaining phenol, mix gently on ice for 10 minutes and place on ice for 10 minutes.
Centrifuge at 3200 rpm for 40 minutes and collect the aqueous layer.
11. Extract with 7 ml Chloroform/Isoamylalcohol (24:1) and mix gently on ice for 10
minutes and place on ice for 10 minutes. Centrifuge at 3200 rpm for 40 minutes,
remove aqueous layer.
6

7. 12. Precipitate DNA by adding one-tenth volume of 10 M ammonium acetate followed by
2 volumes of absolute ethanol (or an equal volume of isopropanol), mix until DNA is
visible. Store at -20°C overnight or at -70°C for 15 minutes.
13. Centrifuge at 3200 rpm for 90 minutes at 4°C, decant supernatant and add 5 ml of 70%
ethanol (tap pellet so it floats) and again centrifuge at 3200 rpm for 40 minutes, decant
supernatant and vacuum dry for 10 minutes. Add 1 ml of 10 mM Tris to resuspend the
DNA. Store at 4°C.
14. Measure optical density (OD) of the sample at 260 nm and 280 nm (ideally 260/280
ratio=1.7-2.0; Ratio >2.0 phenol contamination; ratio< 1.7 protein contamination)
Calculate DNA concentration by following formula:
Abs 260 x Dilution factor x 50= DNA concentration (µg/ml)
(50=correction factor)
Transfer sample to a labeled Eppendorf tube. Store at 4°C.
7

8. EXPERIMENT
LOCALIZATION OF DISEASE GENE FOR INHERITED EYE
DISORDERS BY LINKAGE ANALYSIS
Introduction
Linkage analysis is a powerful method of gene mapping. The primary goal is to determine if two
or more genetic traits – i.e. a marker locus (or multiple marker loci) and a disease trait – are co-
segregating within a pedigree. The results of linkage analyses are typically shown as “LOD
Scores” for a pair of loci at various recombination values. LOD stands for log to base 10 of
odds, and odds are that two loci are linked verses the two loci are not linked. LOD score is
calculated with help of computer programs: MLINK or LIPED which are part of the linkage
analyses package, as described in the Handbook of Human Genetic Linkage.
Equipment and Materials
10X PCR Buffer (Sigma) 100 mM Trizma-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl2;
0.01% (w/v) gelatin
dNTPs 10 mM dATP; 10 mM dCTP; 10 mM dGTP; 10 mM
dTTP
Autoclaved deionized H2O aliquot in 1.5 ml eppendorf tubes
DNA samples 40 ng/ µl
Forward & Reverse Primers 20 µM
Taq Polymerase 2.5 U/µl
10X TBE Trizma base (Tris[hydroxymethyl]aminomethane) 70 g/L
boric acid 55g/L
EDTA (Ethylene diamine tetraacetic acid) 9.04 g/L
check pH (if not 8-8.2, then adjust the pH).
filter through millipore filter paper (0.45 µm).
40% Acrylamide solution Acrylamide 389.6g/L
N,N’-methylone bis-acrylamide 10.4g/L
store in dark bottles at 4oC
Gel loading dye 7.5% Ficoll
0.01% bromophenol blue
0.01% xylene cyanol
25% APS freshly prepared
TEMED
10% ethidium bromide
8

9. Procedure
1. Calculate how much master mix is required for the desired number of reactions.
2. Prepare master mix as given below by adding reagents in sequential order (8 µl X number of
reactions; include 2-3 extra reactions).
master mix final conc. required vol.per reaction
17reactions
Deionized H2O 5.0 µl 85.0µl
10X PCR Buffer 1X 1.0 µl 17µl
2.5 mM dNTPs 100 µ M 0.4 6.8µl
Taq Polymerase (2.5 U/µl) 2.5 U 1.0 µl 17.0µl
20 µM Forward Primer 600 nM 0.3 µl 5.1µl
20 µM Reverse Primer 600 nM 0.3 µl 5.1µl
______________________________________________________
Total 8.0 µl
3. Add 2 µl DNA (40 ng/µl) to each tube.
4. Add 8 µl master mix to each tube. If required add 1-2 drops of mineral oil in each tube to
avoid sample evaporation (not required for PCR machines with heated lids).
5. Set up negative (master mix only) & positive controls (master mix + successfully amplified
DNA containing target sequence).
6. Amplify DNA under the following PCR thermal cycler conditions:
9

10. PCR Conditions
1 cycle of 95°C x 4 minutes
30 cycles of 95°C x 45 seconds
55°C x 45 seconds
72°C x 1 minute
1 cycle of 72°C x 7 minutes
Polyacrylamide Gel Electrophoresis
1. Prepare 8% polyacrylamide gel solution by adding 50 ml of 40% acrylamide solution and
25 ml of 10X TBE q. s. to 250 ml with deionized water.
2. Take 50 ml from the 250 ml of 8% acrylamide solution and add 300 µl of 25% APS and
300 µl of TEMED. Pour into the base of Biorad SequiGen system and allow the gel to
polymerize for 2-3 minutes.
3. To the remaining 200 ml of 8% acrylamide solution, add 850 µl 25%APS solution and
150 µl of TEMED. Pour and allow polymerization for at least 2 hours. Use the
appropriate comb and spacers (0.75 mm).
4. Add 1X TBE buffer (Total volume required = 2 l) in the upper and lower buffer chambers of
the gel unit. Set up the sequencing system and pre-run for 10-15 minutes at 100 watts
constant power.
5 Take out the comb and wash wells with 1X TBE buffer. While taking the comb out care
should be taken, so as not to break the wells.
6 Add 5 µl gel loading dye to amplified product. Load the DNA molecular weight marker
VIII in the first lane of the gel and then load 8 µl of the amplified products in the
appropriate wells.
7. Run the gel at 100 watts for 4-5 hours. (depending up on the size of PCR products). Cut
the gel according to expected band sizes and stain with ethidium bromide (0.5µg/ml final
concentration) for a few minutes.
8. Photograph under UV trans-illumination and analyze the gel photograph to determine
alleles.
9. Calculate the lod score. The results are interpreted as follow:
LOD score Interpretation
0 Linkage or no linkage are equally likely
+ve Possible linkage
-ve Possibly no linkage
+3 or more Linkage
-2 or less No linkage
10

11. LINKAGE ANALYSIS (Flow Chart)
Linkage analyses packageVersion 5.20
Pedigree File Parameters File
By any word processor in ASCII format By program: PREPLINK
At the DOS type PREPLINK and press enter
Column # Descriptions Set all the parameters
1 Pedigree identifier
2 Individual’s ID Save as *.dat
3 Father’s ID ( 0 =founder)
4 Mother’s ID ( 0 =founder)
5 Sex ( 1 =male, 2 =female)
6 Disease ( 1 =normal, 2 =affected)
7 and 7+ Genotype
Save as *.pre
MAKEPED
At DOS MAKEPED infile.PRE outfile.PED
LCP (Linkage Control Program)
file.PED At DOS type LCP
From MAKEPED Enter both files
And Set all the parameters
file.DAT Press Pg Down
From PREPLINK after every entry
Type PEDIN at DOS
LRP (Linkage Report Program)
At DOS type LRP Set all the parameters
Press Pg Down
after every entry
RESULTS
11

12. REFERENCES:
1. Davies KE., Read AP., (1992). Molecular Basis of inherited disease. ED: Rickwood D.,
2nd edition. IRL Press, Oxford.
2. Ott J. (1974). Estimation of the recombination fraction in human pedigree: Efficient
computation of the likelihood for human linkage studies. Am. J. Hum. Genet. 26: 588-597.
3. Lathrop GM., Lalouel JM., Julier C. and Ott J. (1984). Strategies for multilocus
linkage analysis in humans. Proc. Natl. Acad. Sci. USA. 81: 3443-3446.
4. Terwilliger J., OttJ. (1994) Handbook for Human Genetic Linkage. Johns Hopkins
University Press Baltimore, MD
12

13. EXPERIMENT
DETECTION OF MUTATIONS IN INHERITED EYE DISORDERS BY
AUTOMATED FLUORESCENT SEQUENCING
Introduction
Automated sequencing will be performed on an ABI 377 DNA sequencer (Perkin Elmer), using
the ABI PRISM™ Amplitaq® DNA polymerase FS, Dye Terminator Cycle Sequencing Ready
reaction Kit. Automated cycle sequencing has several advantages over manual radioactive
incorporation techniques. Foremost, it is safer as it eliminates the use of radioactive nucleotides.
The technique is based on using 4 fluorescent dye-labelled terminators, which are detected by
lasers that analyse and convert the DNA sequence to a graphical image on a computer. Reactions
are also carried out in single tubes, and do not have to be separated for each terminator
nucleotide. Less starting material is required and the sequencing of larger templates is possible.
For each sequencing reaction one gel lane is utilized, because the labeled nucleotides can be
distinguished by unique fluorescent labels.
Equipment and Material
PCR machine (Hybaid)
PCR Reagents (10X PCR buffer, dNTPs, DNA Taq polymerase, primers).
ABI 377 DNA sequencer (Perkin Elmer).
ABI PRISM™ Amplitaq® DNA polymerase FS, Dye Terminator Cycle Sequencing Ready
reaction Kit.
Qiagen (QIAquick gel extraction kit, cat no. 28706).
95% and 70% ethanol.
Sequence specific primers.
Acrylamide gel (Sequencing Grade).
Gel loading buffer (5:1 formamide/dextran blue).
Procedure
1. Amplify DNA by polymerase chain reaction in 50µl reaction volume. Reaction mixture
include:
Reaction component µl per reaction Final concentration
Sterile H2O 35
10X PCR buffer 5.0 1X
2.5 mM dNTPs 2.0 100µM
DNA Taq polymerase 1.0 2.5 units
Primer forward (20µM) 1.0 1.0µM
Primer reverse (20µM) 1.0 1.0µM
DNA template (10ng/µl) 5.0 50ng
13

14. Amplify under following PCR cycling conditions:
Step1. 950C for 4 minutes 1 cycle
Step2: 950C for 1 minute (denaturation)
Step3: 660C for 1 minute (annealing)
Step4: 720C for 1 minute (extension) repeat step 2-4 for 35 cycles
Step5: 720C for 7 minute (final extension) 1 cycle
2 Purify PCR products by using QIAquick Gel extraction kit (Cat no. 28706) according to
manufacture's instructions, to remove primers and unincorporated dNTPs.
3 Setup sequencing reactions by adding:
Sterile H2O 2.0 µl
Terminator ready reaction mix.
(Includes labelled dye terminators, buffer, and dNTPs) 4.0 µl
Forward or Reverse sequence specific primer 1.0 µl
TemplateDNA (0.5µg) 1.0 µl
Total reaction volume 10.0 µl
4. Subject to 25 cycles of 96oC for 10 seconds, 50oC for 5 seconds and 60oC for 4 minutes in
a Hybaid thermal cycler.
5. Precipitate the DNA to remove excess unincorporated fluorescent dye with 2.5 volume of
95% ethanol in the presence of 0.3 M sodium acetate.
6. Wash with 70% ethanol and vacuum dry.
7. Resuspend pellet in 5µl of ABI loading buffer, and denature samples at 95oC for two
minutes before loading onto the denaturing acrylamide sequencing gel.
14

15. EXPERIMENT
MICROSATELLITE ANALYSIS OF HUMAN POPULATIONS USING
MULTIPLEX PCR
Introduction
Microsatellite DNA consists of short arrays of tandem repeats which are simple in sequence (1-6
bp) and are dispersed throughout the genome. These are classified as mono, di, tri, tetra, penta
and hexanucleotide repeats. These microsatellites provide an insight into the evolutionary
relationship between the human and other related species and among the various human
populations and have been used extensively for linkage analysis.
Equipment and Materials
PCR Machine (Hybaid)
Electronic Multichannel Dispenser (Biohit)
DNA Sequencer (ABI-377)
Acrylamide gel casting equipment
Tips and Pipettes
SuperTaq Polymerase 5U/µl stock
Taqstart Antibody
Antibody Dilution Buffer
10X PCR Buffer
2.5mMdNTPs
25mM MgCl2
20ng/µl DNA samples
10µM fluorescent dye-labeled primers
10X TBE
40% Acrylamide Solution
Loading Dye (Dextran Blue)
Internal Lane Standard (Tamra 350 or 500)
10 % APS solution
TEMED
Urea
10X TBE
Tris-Base 108 g
Boric Acid 55 g
Na2EDTA.2H2O 8.3 g
Dissolve Tris, Boric acid and EDTA in about 800 ml of high grade deionized water, check the
pH which should be 8.2±0.2. Discard the buffer if the pH is not correct, do not try to adjust the
pH. Transfer the buffer to 1 l cylinder, adjust the volume to 1 l with deionized water. Filter
through a 0.2 µm filter and store at room temperature.
15

16. 40% ACRYLAMIDE SOLUTION (100 ml)
Acrylamide 38 g
Bis-Acrylamide 2g
Dissolve acrylamide and bis-acrylamide in enough water to bring volume to 90 ml. Add 10 g of
mixed bed ion-exchange resin and stir at room temperature until all crystals dissolve, then
continue to stir for additional 5-10 minutes and filter through 0.2 µm membrane filter. Transfer
to 100 ml graduate cylinder and adjust volume to 100 ml with deionized water. The solution is
stable for 1 month at 40C.
Procedure
Multiplex PCR for 10 reactions
1. To 0.52 µl antibody buffer, add 0.20 µl TaqStart antibody and 0.016 µl SuperTaq
polymerase and incubate at room temperature for 7 minutes.
2. Prepare primer mixes of 6 to 7 primer pairs by mixing 0.06-0.12 µl of each primer.
3. To the primer mix add 0.6 µl of 10X PCR Buffer, 0.5 µl MgCl2 , 0.72 µl
of each dNTP mix, 0.736 µl of Taq /antibody mix as prepared in step 1 and 1.925
µl water to make up volume to 6 µl.
4. Add 1 µl DNA to PCR tube and 5 µl of mix prepared in step 4 and amplify by
using the following touch down PCR conditions:
14 Cycles of 940C, 20 seconds
630C decreased by 0.50C per cycle, 1 min
720C, 1 minute
20 Cycles of 940C, 20 seconds
560C, 1 minute
720C, 1 minute
1 Cycle of 40C, 10 minutes
5. The PCR product is saved at 40C until it is loaded on the gel.
Gel Electrophoresis
Preparation of working gel solution (4 %)
Urea 5.4 g
40 % Acrlyamide solution 1.5 ml
10 X TBE 1.5 ml
16

17. 1. Disslove 5.4g urea in 5 ml water contained in a 100 ml beaker. Add 1.5 ml 40 %
acrlyamide solution, and 2-3 g mix bed ion-exchange resin and stir for a couple of
minutes. Filter the solution through a Whatmann No.1 filter paper.
2. Add this solution into a 50 ml graduated cylinder containing 1.5 ml 10X TBE and make
up volume to 15 ml. Filter this solution through a 0.2 µm membrane filter using a
Millipore vacuum filtration assembly.
3. To the filtered solution add 75 µl of 10 % APS and 10.5 µl of TEMED just before
pouring the gel.
Gel Pouring
1. Always use absolutely clean plates. Place the rear plate on the gel casting apparatus with
the inside of the plate facing up.
2. Moisten the spacers with water and place on the rear plate. Place the front plate on the
top of the rear plate.
3. Fill the acrylamide solution into a 50 ml syringe, pour slowly onto the rear plate and
simultaneously slide the front plate on the rear plate taking care that no bubbles are
introduced during the process.
4. Insert the flat edge of the comb at its place and leave for 15 minutes. After 15 minutes
remove the comb and wash the plates with deionized water to remove gel or any other
particle present on the outside of the plates. Leave the plates for about 45 minutes before
use.
Electrophoresis
1. Mix 0.3 of the sample with TAMRA350 internal lane size standard and electrophorese on
an ABI377 DNA sequencer for one and a half hours on a 12 cm plate and collect the data
using ABI collection software.
2. Size the samples using GeneScan software and assign allele sizes using GenoTyper software.
17

18. EXPERIMENT
HLA TYPING FOR HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, & DQB1
USING SSP-PCR.
Introduction
The Human Leukocyte Antigen (HLA) genes of the major histocompatibility complex (MHC)
form the most polymorphic genetic system known in humans. It is present on the short arm of
chromosome 6 and makes up about 1% of the human genome. HLA is divided into three major
regions classified as Class I, Class II and Class III. Genes in the Class I and Class II regions
encode highly polymorphic cell surface glycoproteins which are invovled in antigen
presentation to the T-cells during an immune response. The Class I consists of HLA-A, HLA-B
and HLA-C loci. The Class II is divided into DP, DR and DQ regions. Class I and II can be
typed by using the following method.
Equipment and Materials
PCR Machine (Hybaid)
Electronic Multichannel Dispenser (Biohit)
Gel Tank & 4 Combs (40 wells each)
Power Pack (Bio-Rad Power Pac 3000)
Polaroid Photographic System with UV transilluminator (Fotodyne)
96-Well PCR plates (Hybaid)
10X PCR Buffer
20 mM dNTP Mix
1.0 M MgCL2
Taq Polymerase
Agarose Gel
Orange G Loading Dye
10X TAE
Boehringer Marker VIII
Ethidium bromide
10X PCR Buffer
Tris-base (670mM) 40.568 g
(NH4)2SO4 (166mM) 10.96 g
Tween 20 (1 %) 5.0 ml
Dissolve tris in 400 ml sterile deionized water and adjust the pH to 8.8. Add ammonium Sulfate
to the solution and filter through a 0.2 um filter. Add tween 20 and make up the volume to 500
ml. Store at –20oC.
10X TAE ( 0.04M Tris-acetate and 0.001M EDTA)
Tris 48.40 g
18

19. Glacial Acetic Acid 11.42 g
EDTA 0.5 M (pH 8.0) 20 ml
Dissolve in 800 ml deionized water and q. s. to 1 L. Filter through a 0.45µm membrane filter.
Orange G loading dye (6X)
Orange G (0.125%) 50 mg
Ficoll (20%) 8g
0.5 M EDTA (100 mM) 8 ml
Deionized H2O 32 ml
Dissolve Ficoll and Orange G in water and add EDTA to it.
TDMH (12 vials)
10 X PCR Buffer 2.444 ml
20mM dNTP Mix 216 µl (final conc. 460 µM)
1.0 M MgCl2 58.7 µl (final conc. 6.25 mM)
Autoclaved Deionized H2O 6.684 ml
Store at -20oC as 775µl aliquots.
Procedure
1. Prepare the master mix (155 reactions)
TDMH 775 µl
DNA (0.0625 mg/ml) 248 µl (0.1 ng/reaction)
Autoclaved Deionized H2O 199 µl
Taq Polymerase 18 µl (0.1875 units/reaction)
The PCR amplifications are performed in 13 µl final volume in 96 well plates. 5 µl of
each of the 144 primers is aliquoted into 0.2 ml appropriately labeled 96 well PCR plates
using the multichannel dispenser. 8 µl of the master mix is added to each primer.
2. The following conditions are used for the PCR;
1 min denaturation 96oC
5 cycles of 96 oC for 25 seconds
70 oC for 45 seconds
72 oC for 45 seconds
21 cycles of 96 oC for, 25 seconds
65 oC for 50 seconds
72 oC for 45 seconds
4 cycles of 96 oC for 25 seconds
55 oC for 60 seconds
72 oC for 2 minutes
19

20. 1 cycle of 72 oC for 10 minutes
3. Following PCR amplification, the plates are stored at 4 oC until electrophoresis. 5 µl of
the Orange G loading dye is added to each tube and the PCR products areloaded onto a
2% agarose gel prepared in 0.5X TAE and run at 120 V for about an hour or till the dye
migrates at least 3 cm.
4. The gel is stained in ethidium bromide (0.5µg / ml in deionized water) for 1 hour and
photographed under UV light. The positive bands are scored and the haplotype
determined.
20

21. EXPERIMENT
DETECTION OF SRY-8299 G to A POLYMORPHISM USING
RESTRICTION FRAMGMENT LENGTH POLYMORPHISM
TECHNIQUE (PCR-RFLP)
Introduction
SRY-8299 is a single nucleotide polymorphism (SNP) found on Yap+ chromosomes. It is a G to
A transition that results in the destruction of a BsrBI site.
Equipment and Material
PCR Machine (Hybaid)
Gel Tank and Combs
Power Pack (Bio-rad Power Pac 3000)
Polaroid Photographic System with UV Transilluminator (Fotodyne)
96 well PCR Plates
10X PCR Buffer
25mM MgCl2
2mM dNTP mix
10 µM Primer mix
• -8299f 5’-aca gca cat tag ctg gta tga c- 3’
• -8299r 5’-tct ctt tat ggc aag act tac g- 3’
2.5 Units/µl Taq Polymerase
Autoclaved deionized water
20 ng/µl DNA dilution
Agarose gel
10X TAE Buffer
Orange G loading dye
ethidium bromide
10X PCR Buffer
Tris-base (670mM) 40.568 g
(NH4)2SO4 (166mM) 10.96 g
Tween 20 (1 %) 5.0 ml
Dissolve Tris in 400 ml sterile deionized water and adjust the pH to 8.8. Add
ammonium sulfate to the solution and filter through a 0.2 um filter. Add Tween 20
and make up the volume to 500 ml. Store at –20oC.
10X TAE (0.04M Tris-acetate and 0.001M EDTA)
Tris 48.40 g
Glacial acetic acid 11.42 g
EDTA 0.5 M (pH 8.0) 20 ml
Dissolve in 800 ml deionized water and q. s. to 1 L. Filter through a 0.45 µm
membrane filter.
21

22. Orange G loading dye (6X)
Orange G (0.125%) 50 mg
Ficoll (20%) 8g
0.5 M EDTA (100 mM) 8 ml
deionized H2O 32 ml
Procedure
1. Each reaction tube consists of 25 μl final volume. Prepare master mix as follows:
Reagent final conc./reaction μl/reaction
10 x PCR Buffer 1x 2.5
25 mM MgCl2 1.5 mM 1.5
2 mM dNTP mix 200 μM 2.5
10 μM Primer mix 1 μM 2.5
2.5 U/μl Taq Polymerase 1.25U 0.5
Deionized water 13.5
Add 23 μl of the above mix to 2 μl DNA dilution (40 ng/ reaction)
2. Amplify using the following conditions
3 minutes denaturation 94oC
30 cycles of 94 oC for 30 seconds
60 oC for, 30 seconds
72 oC for 60 seconds
1 cycle of 72 oC for 5 minutes
3. Run 10 μl of the PCR product on a 1.5% agarose gel to check for amplification. The
product size is 509 base pairs.
4. BsrBI Digestion
To 15 μl of the amplified SRY-8299 product add 5.8 μl of the following mix
Reagent Final conc./reaction μl/reaction
BsrBI (10 units/ μl) 5 units/reaction 0.5
10X buffer 1X 0.6
Deionized water 4.9
22

23. Digest overnight at 37oC. Run the digested product on a 1.5% agarose gel. Digested
fragments sizes are 147 and 362 base pairs and indicate the presence of the ancestral G
allele. The loss of BsrBI site indicates the presence of the derived A allele.
Lanes 1-6: PCR SRY-8299 digested by BsrBI
Lanes 1,2,4,5: A allele without the BsrBI site
Lanes 3,6: G allele with the BsrBI site
23

24. EXPERIMENT
DETECTION OF SRY-8299 G to A POLYMORPHISM BY DENATURING
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC)
Introduction
The Transgenomic WAVETM DNA fragment analysis system is an exceptionally powerful tool
for mutation detection and size-based DNA fragment analysis. DNA fragment separation is
carried out by use of a specialized DNASep column (Part # 18-621-0546) based upon the
principle of ion-pair reversed phase HPLC. After eluting from the cartridge, the separated DNA
fragments travel to the detector where they are detected by UV absorption.
The system consists of the following instruments stacked on top of each other. The interface,
pump, temperature rack (chiller), auto-sampler, column oven and uv detector. The degasser and
computer system are placed separately (Figure 1). The pump has four inlets (1-4), which are
connected to bottles A (1), B (2), C (3) and D (4), which go directly to the injection valve.
Bottles A and B contain the DNA separations buffers A and B respectively. Bottle C contains
the column wash solution (75% acetonitrile) and bottle D contains the syringe wash solution (8%
acetonitrile).
Equipment and Materials
Transgenomic WAVETM DNA fragment analysis system.
DNA separations buffers A and B
Column wash solution (75% acetonitrile); Buffer C
Syringe wash solution (8% acetonitrile); Buffer D
PCR Machine (Hybaid)
Tube-strips (Hybaid)
Buffer A (0.1 M triethylammonium acetate (TEAA) solution, pH 7.0)
Buffer B (0.1 M TEAA solution containing 25% acetonitrile, pH 7.0)
Buffer C (Column Wash Buffer; 75% acetonitrile solution)
Buffer D (Syringe Wash Buffer; 8% acetonitrile solution)
Procedures
1. To prepare the mutation standard for analysis on the WAVE DNA Fragment Analysis
System, transfer approximately 15 to 20 µl of the WAVE optimized UV 209 bp mutation
standard (Part#560077) to a microtiter tube and keep at 4oC until ready to use. This
standard consists of a combination of two analog 209 bp fragments representing A and G
alleles at position 168 of the polymorphic DYS271 locus. Upon heating and renaturation
this fragment mixture forms two homoduplexes which are used to check instrument
parameters for heteroduplex-based mutation screening.
24

25. 2. Combine equal amounts (10 µl) of the previously amplified 509 bp SRY –8299 wildtype
and mutant fragments in a single tube. Denature the fragments at 95oC for 5 minutes and
carry out heteroduplex annealing by decreasing the temperature by 1.5oC/min until it
reaches 25oC. Store the test samples at 4oC if not used immediately. If a 96-well PCR
plate is used, simply transfer it from the thermal cycler into the WAVE ®system’s
constant temperature rack (Peltier plate) for analysis. This eliminates the requirement for
manual sample loading, allowing unattended sample processing. Alternatively, 0.2 ml
tube strips containing DNA sample can be loaded in the appropriate slots. Carefully
place the tube containing the standard in position 1 at the rear left corner of the 96-well
auto-sampler plate (Figure 2). Place the tube containing the wildtype SRY-8299
fragment in position 2 and the heteroduplex in position 3.
3. Based upon the sequence of the wild type DNA determine the correct temperature for
mutation scanning empirically, or by using the WaveMaker utility software. Final
temperature selection is confirmed experimentally by performing separations at the
calculated temperature and at 20C above and below it using the universal gradient. The final
retention time for the calculated temperature should then be roughly midway between those
obtained at the higher and lower temperatures.
4. Click on the Application button on the Main Tool Bar at the left of the main screen. The
Application dialog appears. Highlight the name of the Transgneomic Application Folder and
click on Select. Confirm that the name of the selection appears on the title bar of the main
screen.
5. Click on the Method Setup button on the main tool bar to open the method for mutation
detection. In the File Type box, select Methods. In the Application list box, verify that the
selected folder is highlighted. In the Method Name box, highlight the name of the method
for e.g Mutation Detection and click OK. A method information window appears in the
display area of the main window.
6. Click the left mouse button on the red pump icon to open the Pump Setup window and enter
the appropriate method gradient as determined empirically or by WAVE MAKER software.
The recommended gradient for mutation detection is a slope of 2% increase in the
concentration of Buffer B per minute. The gradient should be run initially at 50 oC under
non-denaturing conditions at a flow rate of 0.9 ml/min. For an unknown mutations a
universal gradient for mutation detection is shown below:
Time % Buffer A % Buffer B
0 65 35
0.1 60 40
16.1 28 72
16.2 0 100
16.7 0 100
16.8 65 35
18.8 65 35
25

26. 7. At the end of each series of runs program a clean method followed by column
equilibration for 3-5 minutes and a sleep mode as shown below:
Time % Buffer % Buffer % Buffer Flow Rate Method
A B C
18.9 0 0 100 0.9 ml/min Clean
48.9 0 0 100 0.9 ml/min Clean
49.0 50 50 0 0.9 ml/min Equilibrate
54.0 50 50 0 0.9 ml/min Equilibrate
54.1 50 50 0 0.05 ml/min Sleep
8. Click on the Sample Set Up button on the main tool bar to open the Open File dialog
box. In the application list box, verify that the correct folder is selected. In the Sample
Table Name box, highlight the name of the Mutation Detection Sample Table and click
OK. The Setup Information dialog box appears. Make sure that the standard sample is
coming from vial 1, the volume is 5 μl and the number of injections is 1 (Inj per Vial).
Enter an appropriate Vial No. and Vol (μl) to be injected and specify the injections per
vial (Inj per Vial). Enter an appropriate Sample Name and specify the Method Name.
Confirm that the vial number, volume and the number of injections are correct. For each
sample and standard make sure that the Max Ch 1 Noise and Max Ch 1 drift is set at 500.
9. Got to File and click on Save sample As… option and give an appropriate name to the
Sample Table.
10. Click on the auto-sampler Set up (L-7200) icon and select the injection method. The Cut
Method is usually preferred. Make sure the correct syringe volume (500 μl) is selected.
11. Click on the column Oven Setup icon and enter the appropriate temperature for mutation
detection. Do not change the Temperature Upper limit which is set at 80oC.
12. Click on Channel 1 Detector Setup (L-7400) and adjust Data Acquisition Stop Time (min)
and Doubling Time (min) to the method generated. Leave all other settings unchanged and
click on Generate Sampling Period Table.
13. Go to File on the menu bar and select the Save Method As… option and give an appropriate
name to the method file.
14. Click on the blue Initialize button to open the Hardware Status Window and click on
Initialize to make the instrument operative. This may take a few minutes.
15. The Start Series function is available only when the System Manager program is ready to
perform the injections and data acquisition, click the Start Series button at the bottom of the
Data Acquisition window. Before the System Manager program starts the first injection, it
validates the first row in the Sample Table. Also, the column oven must have reached the
correct temperature before Start Series can be performed.
26

27. 16. Pressing the “Start Run” button at the bottom of the Data. Acquisition Monitor window
will perform only a single injection for the first row listed in the Sample Table. Pressing
the “Start Series” button performs the entire series of injections listed in the Sample
Table.
17. To view the sample file created at the end of the run, click the Reprocess Data button.
This brings up the Open file dialog. In the File Type box, select “Data”.
18. Select the last file name in the list (this corresponds to the last series of runs performed)
and click Ok. This brings up the Injection Table for the series. Select (highlight) the first
chromatorgram listed in the Injection Table and click the Display button at the bottom of
the window to display the data.
19. To adjust the view, click the Chromatogram Display Options button in the horizontal
tool bar, or double-click anywhere in the display are of the screen.
20. When the instrument is not in use, it is important to keep the pump running at a very low
flow rate. The column can be maintained on the instrument for two to four days by pumping
the column at a flow rate of 0.05-0.1 ml/min with 50% Buffer A, 50% Buffer B.
27

28. Figure 1. Homo (upper panel) and hetero-duplexes (lower panel ) showing the SRY-8299
ancestral (G) and derived (A) alleles respectively.
28